Type 2 diabetes (T2D) is a prevalent disease affecting a large population worldwide. Emerging evidence suggests that impaired muscle metabolism plays a major role in the pathogenesis of T2D. Normalizing mitochondrial function has be proposed as an effective therapeutic target for T2D. Phophorus-31 (31P) magnetic resonance spectroscopy and imaging (MRS/I) methods provide a powerful tool to interrogate various aspects of tissue metabolism. Specifically, 31P MRS can directly monitor changes in phosphate metabolite concentrations during physiological and pathological processes such as exercise and ischemia/reperfusion. Furthermore, magnetization-transfer (MT) methods allows noninvasive quantification of metabolic activities that no other methods are capable of. These methods have the potential to be translated to clinical use that will permit noninvasive evaluation of tissue metabolism that is indicative of disease progression and therapeutic efficacy. However, because of the low concentrations of phosphate metabolites, current 31P MRS/I methods require prohibitively long acquisition time, which is not practical for routine clinical use. Consequently, most 31P MRS studies have employed either non-localized or single voxel techniques, rendering the assessment of metabolic heterogeneity impossible. Recent development in sparse sampling theory and subspace imaging have demonstrated potential by significantly reducing acquisition time for proton (1H) MRSI. Furthermore, the innovation brought forth by magnetic resonance fingerprinting (MRF) has been shown to drastically accelerate the mapping of 1H relaxation times in the brain. Based on these exciting progress, we propose to translate these 1H MRSI approaches to develop clinically feasible 31P MRS/I methods for in vivo assessment of mitochondrial function in diabetic muscle. The objectives of the proposed project are: 1) to develop 31P spatiospectral encoding method with sparse sampling of the (k, t)-space for highly accelerated metabolic mapping of phosphate metabolites; 2) to develop 31P spectroscopic MT-MRF methods for efficient quantification of ATP and PCr synthesis rates. These methods will be applied to delineate the alterations in metabolic fluxes and mitochondrial oxidative capacity in Zucker diabetic fatty rats, a rat model of obesity and insulin resistance. In addition, the effects of exercise training and metformin treatment, the commonly prescribed treatment for T2D, on muscle metabolism will also be evaluated. The successful completion of this project will pave the way for evaluating mitochondrial energetics in vivo. While the current project will employ a rodent model of T2D for the purpose of cost-saving, the proposed 31P MRSI and MRF methods are highly translatable to clinical scanners, which will lead to clinically feasible and relevant diagnostics, as well as novel strategies to assess the therapeutic efficacy for a spectrum of metabolic diseases.